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European Biopharmaceutical Review

Measurable Advances


Novel fibre optic chemical sensors allow researchers to monitor DO and pH levels in real time and without disturbing a sealed environment, offering significant benefits when analysing bioreactor processes.


Advances in high-performance sensor materials and optoelectronics have enabled the development of novel optical sensors for use in applications in life sciences, biopharmaceuticals, biotechnology, food and beverage processing and more. Compared with traditional electrochemical sensing techniques such as galvanic sensors, optical sensors can be made in small and customisable form factors, allow non-intrusive measurements and do not consume the sample (see Table 1). The principle of operation is to trap an oxygen-sensitive fluorophore or pH indicator dye in a sol gel host matrix that can be applied to the tip of a fibre, an adhesive membrane such as a patch, or a flat substrate such as a cuvette or microtiter plate.



The indicator materials change optical properties in response to specific analytes in their immediate environment and electronics, then measure the response.

For oxygen, a phase fluorometer measures the partial pressure of dissolved or gaseous oxygen; for pH, a miniature fibre optic spectrometer measures the colorimetric (absorbance) response of the pH dye. In pH sensing, the surface is a nanoporous material formulated to trap pH indicator molecules in a cage-like structure. The cage is hydrophilic, so hydronium ions freely diffuse through the film. The trapped bromocresol green ions change colour when they bind or dissociate with hydronium ions. As a result, an optical pH sensor can be embedded anywhere in the process stream, free from the handling problems associated with electrodes and other pH measurement technologies.

Fibre Optic Chemical Sensors

Oxygen optrodes are relatively new compared with electrochemical sensors. As chemistries and detector electronics improve, the use of optrode sensors is increasing. Most optrodes use a polymer-based host matrix to trap the fluorophore. Polymer-based oxygen optrodes have the advantage that they can be produced in bulk, with excellent lot-to-lot repeatability for calibration consistency. The main disadvantage is that the electronic environment of the polymers promotes photodegradation of the fluorophore, so the overall operating lifetime is limited.

Another host matrix for oxygen sensing uses sol gels. Introduced in the 1990s, sol gels are a nanoporous, glass-like material that typically consists of alkoxide metal molecules that have been mixed with water and a solvent to create a homogeneous, partiallypolymerised liquid called a sol (1,2). The fluorophore is dissolved in the sol, which is then applied to the substrate host (fibre tip or flat substrate) as a thin film, dried and cured to a hardened glass-like substance. Sol gels are widely used in many manufactured products as protective coatings and are also easy to manufacture in high volume.

Other sol gel materials have been developed to encapsulate pH dyes for optical pH sensing. These materials can be applied to various sampling devices – including cuvettes, optical fibres and probes – and can be combined with miniature fibre optic spectrometers and accessories for convenient, nonintrusive monitoring (see Figure 1). What’s more, pH-sensitive transducer materials can be formulated as selfadhesive patches that can be integrated into biological and other types of sample container.



One disadvantage of optical pH sensor products based on colorimetric transmissive measurements is their susceptibility to ambient light interferences and sample colour or turbidity effects. Newer approaches employ electroformed mesh to metalise the pH sensor material, providing an optically reflective yet ion permeable membrane between the sensor and the environment. The electroforming process creates highly precise mesh sheets through atomby- atom electrodeposition in a plating bath. The hole and wire sizes that can be achieved are of the order of a few microns, with sub-micron repeatability. This novel technique is ideal for colorimetric sensor applications.

One consideration in evaluating the benefit of optical and pH sensors for use in biopharmaceutical processes is the biocompatibility of the sensing materials. For example, in the US, most manufacturers require that various sensing material form factors meet United States Pharmacopeia (USP) Class VI certification standards (ISO and European Pharmacopoeia standards may apply in other jurisdictions). The USP is a non-governmental standard-setting organisation. Class VI is its most stringent testing protocol for classification of plastics used in medicines and other health care technologies. With USP Class VI certification, the biocompatibility, toxicity and extractables of sensor coatings are assured to be compatible with biological and pharmaceutical processes and implantable devices.

Experimental Conditions

To demonstrate the viability of optical oxygen and pH sensors for monitoring biological parameters in bioreactor environments, we placed oxygen- and pHsensitive adhesive patches inside a bioflask, in which red grape biofermentation occurred. Bioreactors are closed-environment systems where the cells are cultured under specific conditions to synthesise the final product. Such systems require constant monitoring of DO and pH to optimise bioprocesses. The patches provided oxygen and pH measurements in both headspace and the liquid phase.

Oxygen-sensitive patches were attached to the container with adhesive backing to monitor the oxygen in headspace and in solution. The pH patches were placed in solution to monitor pH changes during the fermentation process (see Figure 2).



Fresh red table grapes (not the type one would typically associate with wine-making) were mashed and the must was left untouched for 48 hours. The juice was drained from the mixture and placed in a bioflask. Yeast cells and nutrients were added to begin fermentation. Non-intrusive oxygen and pH measurements during the aerobic and anaerobic processes were monitored over a 60-hour period.

To measure oxygen, two phase fluorometers were used, both equipped with bifurcated optical fibres for the excitation and detection of the oxygensensitive patches. A phase fluorometer measures the phase shift between a blue LED used to excite the oxygen indicator in the patch and the emission signal of the fluorescence (see Figure 3). The fibres were situated along the outside surface of the flask, pointing directly at the patches on the inside.



One oxygen-sensitive patch was placed in the headspace area of the bioflask. Headspace monitoring is a particular concern within biopharmaceutical and pharmaceutical manufacturing, where careful monitoring of oxygen is related to product discoloration, dissolution rate changes and reduction in shelf life. Parenteral containers, such as blood bags and vials, require careful monitoring of headspace oxygen levels.

To measure pH, a handheld miniature spectrometer with onboard microprocessor was employed, integrated into a vertical stack with a tungsten halogen light source. A bifurcated optical fibre completed the set-up. One leg of the fibre transmitted light to the patch inside the container and the other leg of the fibre read the response from the reflective patch inside the solution. Standard pH buffers were used for calibration. Absorbance curves were observed over time (see Figure 4).



Results

In the first two hours of fermentation, the oxygen sensor patch in the solution detected a quick drop from air saturation as soon as yeast cells and nutrients were added (see Figure 5). The yeast cells had started consuming the oxygen through the liquid cell membrane interface by the diffusion process. The pH sensor in the solution measured a slight drop in absorbance as the oxygen decreased and CO2 was released. The same experiment can be extended to a single cell in a microfluidics well culture system.



Headspace remained at air saturation for approximately the first 2.5 hours of fermentation. Once the oxygen in the solution was completely quenched, the yeast cells and nutrients started consuming the oxygen from the headspace (see Figure 6).



Conclusion

The limitations of electrochemical-based oxygen and pH sensing are overcome by optical oxygen and pH patches. Such patches can be integrated easily within a small-scale biosystem such as a bioflask and provide continuous, non-intrusive monitoring of key system parameters. The ability to monitor DO and pH in real time without perturbing a sealed environment can lead to an improved understanding of the processes in the bioreactor and, ultimately, help to facilitate the development of new biopharmaceutical products and processes.

As a result, in the biopharmaceuticals arena, it can be seen that the use of nonintrusive optical oxygen and pH sensors has implications for applications such as headspace monitoring of parenteral packaging used for biotech drugs, shelf life studies and drug development.

References

  1. Wang W, Reimers CE, Shahriari MR and Wainright SC, The Marine Technology Society Annual Conference Proceedings 1, 1998
  2. Krihak M, Murtagh M and Shahriari MR, Chemical, Biochemical and Environmental Fibre Sensors VIII, Proc SPIE 2836, 1996



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Rob Morris is Director of Marketing for Ocean Optics. Rob joined Ocean Optics as a sales/marketing associate in 1995, during one of the fastest periods of growth in company history, and has been part of the company as it expanded into a worldwide entity with seven locations across the globe. Rob has nearly 30 years of experience in public relations, sales and marketing, primarily in technology businesses. Email: rob.morris@oceanoptics.com

Mahmoud Shahriari is a pioneer in the development of fibre optic chemical sensors and has been a sensors scientist and consultant for Ocean Optics since 1999. Prior to joining Ocean Optics Mahmoud was a Professor of Ceramic and Materials Engineering at Rutgers University. During his tenure at Rutgers, Mahmoud developed several groundbreaking technologies, which resulted in about 70 refereed publications and a number of patents. He holds a PhD in Chemical Engineering and Materials Science and an MSc in Chemical Engineering from The Catholic University of America. Email: mahmoud.shahriari@oceanoptics.com

Harish Dabhi is a Sensors R&D Engineer for Ocean Optics. Harish has an MSc in Electrical Engineering and Management from the University of South Florida. As part of the sensors team at Ocean Optics since 2006, Dabhi has focused on the development, calibration and production of the company’s line of optical oxygen and pH sensors. Email: harish.dabhi@oceanoptics.com

Derek Guenther is a Sensors R&D Engineer for Ocean Optics. Derek has a BSc in Chemical Engineering (with a minor in Chemistry) from the University of South Florida (USF). He worked for three years at the Institute for Environmental Studies at USF as an Environmental Chemist, developing algaecides targeting invasive species in Florida fresh waterways. At Ocean Optics he has helped to develop and support the company’s optical pH sensor products, including probes, patches and other substrates. Email: derek.guenther@oceanoptics.com
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Rob Morris
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Mahmoud Shahriari
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Harish Dabhi
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Derek Guenther
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